The present invention relates to an apparatus for multiphase aqueous separation and more specifically to an apparatus for multistage, countercurrent (sequential) two-phase separation, and is particularly useful for partitioning specific target microorganisms from background material and organisms.
Traditionally, multiphase liquid separation is a labor intensive, hand-operated, wet-lab procedure carried out in laboratory glassware or the like. The extraction procedures are routinely adapted and utilized to extract and separate various reagents, compounds, particles, or other desired samples from their background liquids. The extraction liquids may be any combination of liquids which due to their thermodynamic or solubility properties are able to separate into phases. Typically, extraction by these techniques utilizes two phases for separation, however, a particular extraction protocol could be designed to utilize three or even more phases. Further, the procedure is routinely practiced repetitively, wherein a portion or all of a phase containing the desired sample from one stage is added as the sample to a next stage containing fresh extraction liquids. Thus, the procedure generally can be practiced as multiphase, utilizing two or more phases; and as multistage, wherein the procedure is repeated to enhance the purity of the extracted samples.
In the present system, Applicants have embodied the apparatus of the invention to demonstrate the extraction of target microbial cells from background cells and other materials. A two phase, three stage system is demonstrated utilizing the aqueous polymer phases of polyethylene glycol polyethylene glycol (PEG) and dextran (DEX), which are known in the art to have wide application for the separation of biological materials. Advantages of the disclosed apparatus in this context include increased handling efficiency, reliability, and safety to laboratory workers, especially when contact with the phase liquids or microbial samples is potentially harmful.
A microbiological practice based upon PEG and DEX is described in Walter, H., et al., "Partitioning in Aqueous Two-Phase Systems" (Academic Press, 1985) and Albertsson, "Partitioning of Cell Particles and Macromolecules", 3rd edition (Wiley, 1986). The latter publication describes apparatus for countercurrent distribution of organic matter in phase separation. This apparatus is characterized by complexity and high cost, and requires cleaning between runs to insure absence of contamination.
Strom, in International Application WO 90/05768, describes large-scale industrial apparatus for two-phase polymeric separation of lubricating agents from bacterial contaminants. Only a single-separation, bottom phase accumulation of bacterially contaminated solution is described.